Rho GTPases activated by upstream signals coordinate the cytoskeletal dynamics necessary for such complex motiJe processes as chemotaxis, metastasis, axon guidance, etc. One critical mechanism by which Rho GTPases control actin dynamics is via regulation of the action of cofitin/ADF family proteins. Cofilin/ADF (CtA) proteins act to sever F-actin filaments and promote F-actin depolymedzation, both activities that are critical to the cytoskeletat rearrangements involved in formation of tending edge lamellae and cetl motility. The action of C/A is regulated by phosphoryMation/dephosphoryiation of a criticam regulatory site at Set3. We have previously shown that Rac and Cdc42 regulate the phosphorytation and inactivation of ;/A through the action of a downstream kinase cascade involving p21-activated kinase (PAK) and LIM kinase. We have now identified a unique phosphatase that acts to dephosphorylate and thus activate C/A. in combination with biochemical studies, we will utilize genetic means and RNA interference methodologies to disrupt phosphatase function in vivo. We propose to investigate the properties and regulation of this novel CA phosphatase in order to determine its importance in modulating cytoskeletat dynamic behavior We have discovered regulatory interactions between Ser3-phosphoCA and 14-3-3 family proteins. We will investigate the hypothesis that, in addition to direct regulatory effects on CA, 14-3-3 proteins act as intermolecuiar scaffolds to link CA regulatory kinases and phosphatases into a coordinated regulatory module. Finally, we will investigate the coordinated action of the PAK1-LIMK-CA phosphatase-CA pathway during stimulated cell motility. Regulatory events modulating this signaling pathway will be determined. The question of localized vs. global regulation of CA function wilt be addressed by real time studies of the spatial and temporal dynamics of the signaling molecules involved. The proposed studies shoutd give us unique information about the action of Rho GTPases to regulate CA function during cetl motility.